Inhibition of N- and P/Q-type Ca 2+ channels by cannabinoid receptors in single cerebrocortical nerve terminals

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1 FEBS Letters 579 (2005) FEBS Inhibition of N- and P/Q-type Ca 2+ channels by cannabinoid receptors in single cerebrocortical nerve terminals M.C. Godino, M. Torres, J. Sánchez-Prieto * Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain Received 28 October 2004; revised 15 December 2004; accepted 21 December 2004 Available online 12 January 2005 Edited by Maurice Montal Abstract Since cannabinoid receptors inhibit excitatory synaptic transmission by reducing glutamate release, we have examined whether this might occur through the direct inhibition of presynaptic Ca 2+ channels. In cerebrocortical nerve terminals, activation of cannabinoid receptors with WIN55,212-2 reduces the KCl-evoked release of glutamate. However, this inhibition is attenuated when N- and P/Q-type Ca 2+ channels are blocked. Through Ca 2+ imaging in single nerve terminals, we found that WIN55,212-2 reduced the influx of Ca 2+ both in nerve terminals that contain N-type Ca 2+ channels and those that contain P/Qtype Ca 2+ channels. Thus, cannabinoid receptors modulate the two major Ca 2+ channels coupled to glutamate release in the cerebral cortex. Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Cannabinoid; Glutamate release; Ca 2+ channel; Ca 2+ imaging in single nerve terminal 1. Introduction Endocannabinoids released by postsynaptic cells inhibit neurotransmitter release by activating presynaptic cannabinoid receptors in many synapses, including those in the cerebral cortex [1 6]. It has been suggested that inhibition of presynaptic Ca 2+ channels is responsible for the reduction of glutamate release by cannabinoid receptors. However, it has not been clearly established which types of Ca 2+ channel can be modulated by this presynaptic mechanism. In cultured rat hippocampal neurons, cannabinoids inhibit somatic N- and P/Q-type calcium channels [7]. However, studies of synaptic transmission have indicated a selective inhibition of N-type Ca 2+ channels, since blockage of these Ca 2+ channels subtype with toxins suppresses the response to cannabinoids [1,8,9]. Direct measurement of the effects of cannabinoid on presynaptic Ca 2+ channels has been limited to synapses with a large presynaptic bouton like those at the Calyx of Held [10]. However, monitoring the presynaptic Ca 2+ influx and measuring the postsynaptic current amplitude in brain slices reflect the activation of many synaptic terminals that may or may not express the presynaptic receptor under study [11]. Here, we have used a preparation of cerebrocortical nerve terminals to determine the effect that cannabinoids have on glutamate release. In * Corresponding author. Fax: address: jsprieto@vet.ucm.es (J. Sánchez-Prieto). addition, by imaging Ca 2+ in individual nerve terminals we determined the impact that the activation of cannabinoid receptors has on the Ca 2+ response evoked by depolarization, identifying the type of Ca 2+ channel involved. The results show that cannabinoid receptors reduce the depolarization-induced influx of Ca 2+ in nerve terminals that contain N- or P/Q-type of Ca 2+ channels. As a consequence, they reduce the glutamate release component associated with the activation of these two Ca 2+ channels. 2. Materials and methods 2.1. Synaptosomal preparation The cerebral cortex was isolated from adult male Wistar rats (2 3 months) and the synaptosomes were purified as described previously [12,13] on discontinuous Percoll gradients (Amersham Pharmacia Biotech, Uppsala, Sweden). Following the final centrifugation at g for 10 min, the synaptosomes were resuspended in 8 ml of HEPES buffer medium (HBM): 140 mm NaCl, 5 mm KCl, 5 mm NaHCO 3, 1.2 mm NaH 2 PO 4, 1 mm MgCl 2, 10 mm glucose and 10 mm HEPES, ph 7.4. The protein content was determined by the Biuret method, and 1 mg of the synaptosomal suspension was diluted in 8 ml of HBM and spun at 3000 g for 10 min. The supernatant was discarded and the pellet containing the synaptosomes was stored on ice. Under these conditions, the synaptosomes remain fully viable for at least 4 6 h, as judged by the extent of KCl evoked glutamate release Glutamate release Glutamate release was assayed by on-line fluorimetry. Synaptosomal pellets were resuspended in HBM (0.67 mg/ml) and preincubated at 37 C for 1 h in the presence of 16 lm bovine serum albumin (BSA). The BSA served to bind any free fatty acids released from synaptosomes during the preincubation. A 1 ml aliquot was transferred to a stirred cuvette and the release of glutamate was measured as previously described [14]. The Ca 2+ -dependent release was calculated by subtracting the release obtained during a 5 min period of depolarization in the presence of 200 nm free [Ca 2+ ] from the release at 1.33 mm CaCl Plasma membrane potential The plasma membrane potential was determined with 3,3 0 -dipropylthiadicarbocyanine iodide, DiSC 3 (5). Synaptosomes were resuspended (0.67 mg/ml) in HBM without BSA, and 1 ml aliquots were transferred to a stirred cuvette containing 5 lm DiSC 3 (5) and 1.33 mm CaCl 2. After allowing the mixture to equilibrate for 1 min, the fluorescence was determined at 651 and 675 nm. Data were collected at 1 s intervals Ca 2+ imaging of the responses in single synaptosomes Synaptosomes in HBM (2 mg/ml) with 16 lm BSA were preincubated with 5 lm fura-2 AM and 1.33 mm CaCl 2 for 40 min and the synaptosomal suspension was then attached to a polylysine-coated coverslip for another hour. Synaptosomes were illuminated alternately at 340 and 380 nm for 0.8 s through a 100 objective with the aid of a monochromator (Kinetic Imaging, Ltd., UK). The fluorescence emitted from the nerve terminals was collected through a band-pass filter /$30.00 Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi: /j.febslet

2 M.C. Godino et al. / FEBS Letters 579 (2005) centered at 510 nm and images were obtained using a slow-scan CCD camera (Hamamatsu C4880) operating at 12-bit digitalization (4096 levels). The output from the camera was stored by the computerized imaging system Lucida 3.0 (Kinetic Imaging, Ltd.) and Ca 2+ images were analyzed as described previously [15]. To measure [Ca 2+ ] cyt, synaptosomes were stimulated by 10 s pulses of 30 mm KCl (a bar in each graph) in the presence or absence of pharmacological agonists or Ca 2+ channels toxins. 3. Results 3.1. WIN55,212-2 reduces the glutamate release components associated with N- and P/Q-type Ca 2+ channels Depolarization of nerve terminals with KCl opens Ca 2+ channels and initiates the exocytotic release of glutamate. The Ca 2+ -dependent release of glutamate after 5-min depolarization with KCl was 3.66 ± 0.16 nmol/mg of protein (n = 5; Fig. 1A). The prior addition of the cannabinoid receptor agonist WIN55,212-2 reduced the release by 37.4 ± 4.3% (n = 4; Fig. 1A). The inhibition by WIN55,212-2 of the KCl-induced release was almost completely abolished (2.4% ± 3.5, n =3, data not shown) by the cannabinoid receptor antagonist AM281 (1 lm). Treatment of synaptosomes with pertussis toxin abolished (0.56 ± 5.5%, n = 3, data not shown) the inhibition of release by WIN55, The blockade of Na + channels with tetrodotoxin (TTx) did not alter (37.1 ± 2.1%, n = 3, Fig. 1A) the inhibition of release by cannabinoids excluding the modulation of voltage dependent K + channels associated to action potentials. Furthermore, depolarization of the synaptosomal plasma membrane measured with a membrane potential-sensitive cationic cyanide dye was not modified by WIN55, Thus, the increase in fluorescence induced by 30 mm KCl (57.0 ± 2.4 arbitrary units) was not altered by WIN55,212-2 (55.1 ± 4.3 a.u., n = 5, data not shown). In order to know to what extent this inhibition resulted from a reduction in the activity of Ca 2+ channels coupled to glutamate exocytosis, we performed experiments in the presence of Ca 2+ channel toxins. In cerebrocortical nerve terminals, glutamate release is primarily coupled to Ca 2+ entry through both N- and P/Q-type Ca 2+ channels [15,16], channels that can be selectively blocked by x-cgtx-gvia [17] and x-aga-iva [18], respectively. Blocking N-type channels with x-cgtx-gvia reduced the KCl-evoked release by 30.8 ± 2.6% (n = 3) and partially occluded the inhibition by WIN55,212-2 (17.2 ± 3.8%, n =5,Fig. 1A and B). Blocking P/Q-type Ca 2+ channels with x-aga-iva reduced glutamate release by 65.5 ± 1.3% (n = 4, Fig. 1A and B) and also partially occluded the inhibition of release by WIN55,212-2 in this case by 20.9 ± 2.0% (n = 4; Fig. 1B). Thus, these data indicate that cannabinoid receptors inhibit glutamate release coupled to both the P/Q- and the N-type Ca 2+ channels WIN55,212-2 reduces the entry of Ca 2+ in nerve terminals containing N- or P/Q-type Ca 2+ channels To further examine whether cannabinoid receptors reduce the activity of Ca 2+ channels, we visualized Ca 2+ in single nerve terminals. We have previously shown that the two major subpopulations of cerebrocortical nerve terminals exist, containing N- or P/Q-types of Ca 2+ channels [19]. In these experiments, nerve terminals were first stimulated with KCl in the presence of either x-cgtx-gvia to block N-type channels or x-aga-iva to block P/Q-type channels. Subsequently, KCl was added in the presence of the cannabinoid agonist WIN55,212-2 and finally KCl was added alone. In the experiments with x-cgtx-gvia and WIN55,212-2 (Fig. 2), a total of 812 nerve terminals from five fields were analyzed. In a subpopulation of nerve terminals (9.3 ± 0.5%), the Ca 2+ responses were reduced by x-cgtx-gvia but not by WIN55,212-2, indicating the presence of N-type channels but the absence of WIN55,212-2-sensitive receptors modulating Ca 2+ entry. In another subpopulation of nerve terminals (22.5 ± 1.4%), Ca 2+ responses were insensitive to x-cgtx-gvia but they were reduced by exposure to WIN55,212-2, indicating the presence of WIN55,212-sensitive cannabinoid receptors but not of Fig. 1. WIN55,212-2 reduces the release components associated with N- and P/Q-type Ca 2+ channels. (A) Ca 2+ -dependent release of glutamate evoked by 30 mm KCl in the presence and absence (control) of 5 lm WIN55,212-2 (WIN), 200 nm x-aga-iva (x-aga-iva) or 2 lm x-cgt-gvia (x-cgt-gvia). WIN55,212-2 and the Ca 2+ channel toxins were added 5 and 1 min prior to depolarization with 30 mm KCl. (B) Bar diagrams show the Ca 2+ -dependent release of glutamate 5 min after depolarization with 30 mm KCl in the presence and absence (Control) of the Ca 2+ -channels toxins x-cgt-gvia and x-aga-iva, both in the presence and absence of WIN55,212-2 (5 lm). In the experiments with tetrodotoxin (TTX), the Na + channel blocker (1 lm) was added 1 min prior to depolarization with 30 mm KCl. The results are means ± SEM of 3 5 experiments obtained from the same number of synaptosomal preparations.

3 770 M.C. Godino et al. / FEBS Letters 579 (2005) Fig. 2. Ca 2+ responses are reduced by both x-cgt-gvia and WIN55,212-2 in a subpopulation of nerve terminals. Synaptosomes were fixed onto polylysine coated coverslips and loaded with fura-2 as indicated in Section 2. (A) Representative field of fura-2-loaded synaptosomes under basal conditions at 380 nm. Ca 2+ responses induced by a 10 s application of 30 mm KCl were determined in the presence of 2 lm x-cgt-gvia, (x-cgt- GVIA+KCl) and in the presence of 10 lm WIN55,212-2 (WIN + KCl). Control Ca 2+ responses were induced by 30 mm KCl in the absence of toxins (KCl) at the end of the experiment. Individual responses of the subpopulation of nerve terminals that only responded to x-cgt-gvia (B; n = 3), WIN55,212-2 (C; n = 3) or to both x-cgt-gvia and WIN55,212-2 (D; n = 3). (E) Individual responses of the subpopulation of nerve terminals that did not respond to either x-cgt-gvia or WIN55,212-2 (n = 5). The results are means ± SEM and (n) indicates the number of individual responses. The disc diagram inserted in black indicates the % of nerve terminals showing a given response. N-type Ca 2+ channels. In a third subpopulation of terminals (28.8% ± 4.5), Ca 2+ responses were reduced both by x-cgtx- GVIA and WIN55,212 revealing the presence of cannabinoid receptors and N-type Ca 2+ channels in individual nerve terminals. Finally, 39.0 ± 2.3% of the synaptic boutons were insensitive to both x-cgtx-gvia and WIN55,212. In another set of experiments, nerve terminals were stimulated in the presence of x-aga-iva to block P/Q-type Ca 2+ channels. A total of 455 nerve terminals from four fields were analyzed. This identified nerve terminals whose Ca 2+ responses were inhibited only by x-aga-iva (36.7 ± 10.8%) and another subpopulation of nerve terminals in which the Ca 2+ responses were only reduced by WIN55,212-2 (27.2 ± 4.1%). Terminals in which Ca 2+ entry was blocked both by x-aga-iva and WIN55,212-2, and in which both P/Q-type Ca 2+ channels and cannabinoid receptors are therefore present represented 27.0 ± 3.5% of the total population. Finally, in some nerve terminals (8.8 ± 2.2%) Ca 2+ responses were insensitive to x-aga- IVA and to WIN55, Discussion In this study, we show that the activation of cannabinoid receptors inhibits glutamate release in the whole population of cerebrocortical nerve terminals and that this effect can be partially occluded by blocking N- and P/Q-types of Ca 2+ channels. Ca 2+ imaging experiments of individual nerve terminals indicate that cannabinoid receptors reduce the influx of Ca 2+ in a subset of nerve terminals that express either N- or P/Q-

4 M.C. Godino et al. / FEBS Letters 579 (2005) types of Ca 2+ channels. This suggests that the mechanism by which cannabinoid receptors inhibit glutamate release in the cerebral cortex may involve the inhibition of the two major Ca 2+ channels that trigger release. A number of studies have suggested that blocking Ca 2+ channels is responsible for the inhibition of synaptic transmission by cannabinoid receptors, but only a few studies have directly measured the activity of presynaptic channels [10,11]. We have found that the inhibition of glutamate release by cannabinoid receptors is partially occluded by blocking N- and P/Q-type Ca 2+ channels with x-cgtx-gvia and x- Aga-IVA, respectively. This suggests that cannabinoid receptors reduce Ca 2+ channel activity in some but not in all nerve terminals expressing N- or P/Q-type Ca 2+ channels. We corroborated this proposal by imaging Ca 2+ in synaptosomes that were blocked by x-cgtx-gvia or x-aga-iva and that were sensitive or insensitive to WIN55, These experiments provide evidence that the Ca 2+ channels were directly modulated by cannabinoid receptors. Cannabinoid receptors also modulate K + channels and thus, they can indirectly reduce Ca 2+ channels activity and the subsequent release of glutamate [20]. Nevertheless, it is unlikely that K + channels are activated during KCl-evoked responses in nerve terminals due to the high external K + concentration. In addition, KCl-evoked responses are insensitive to the Na + -channels blocker tetrodotoxin [19,21] and, therefore, it seems more likely that the inhibitory receptors target release-coupled Ca 2+ channels rather than ionic channels involved in the waveform of action potentials. The N- and P/Q-types of Ca 2+ channels are segregated among different populations of nerve terminals in the cerebral cortex of adult rats [20]. The finding that cannabinoid receptors inhibit both types of Ca 2+ channels suggests that they influence the activity of those Ca 2+ channels with which they are co-expressed rather than selectively acting on the signaling pathways specific to a subtype of Ca 2+ channels. Similar results were obtained in a recent study in the cerebellum where the three types of Ca 2+ channels (N, P/Q and R) present in granule cell presynaptic boutons were inhibited by cannabinoid receptors [11]. In contrast, our results do not agree with the selective regulation of N-type Ca 2+ channels by cannabinoid receptors in the hippocampus [8], striatal neurons [1] and in trigeminal caudal neurons [9]. In these preparations, cannabinoid-mediated inhibition of excitatory synaptic transmission was eliminated by pre-treatment with antagonists of N-type Ca 2+ channels. Cannabinoid agonists inhibit the Ca 2+ -dependent release by ± 1.3%, whereas Ca 2+ imaging experiments show that cannabinoid receptors reduced the influx of Ca 2+ in 51 54% of the nerve terminals. If cannabinoid receptors were restricted to glutamatergic nerve terminals these data would indicate that cannabinoid receptors do not exert complete control over glutamate release, in spite of the significant reduction of Ca 2+ influx (Figs. 2C and 3C). However, this possibility is rather unlikely given the high local [Ca 2+ ] c that triggers release [22], since a reduction in Ca 2+ entry would prevent the firing of those nerve terminals expressing the receptor. Alternatively, Fig. 3. Ca 2+ responses are reduced both by x-aga-iva and WIN55,212-2 in a subpopulation of nerve terminals. Ca 2+ responses induced by 30 mm KCl were determined in the presence of 200 nm x-aga-iva (x-aga-iva + KCl) and in the presence of 10 lm WIN55,212-2 (WIN + KCl). Control Ca 2+ responses were induced by 30 mm KCl in the absence of toxins at the end of the experiment (KCl). Individual responses of the subpopulation of nerve terminals that only responded to x-aga-iva (A; n = 6), to WIN55,212-2 (B; n = 8), or to both x-aga-iva and WIN55,212-2 (C; n = 11). (D) Individual responses of the subpopulation of nerve terminals that did not respond to either x-aga-iva or WIN55,212-2 (n = 8). The results are means ± SEM and (n) indicates the number of individual responses. The disc diagram inserted in black indicates the % of nerve terminals showing a given response.

5 772 M.C. Godino et al. / FEBS Letters 579 (2005) imaging may detect the effects of cannabinoids on GABAergic nerve terminals as well as on glutamatergic nerve terminals explaining the increased sensitivity of this technique. We have previously shown that GABAergic nerve terminals represent a fraction of 24% in the cerebrocortical preparation [20]. Furthermore, cannabinoid receptors are known to be expressed in GABAergic nerve terminals where they reduce the release of GABA by a mechanism that involves the inhibition of Ca 2+ channel activity [23]. In summary, we show that the two major types of Ca 2+ channels coupled to glutamate release in the cerebral cortex are modulated by cannabinoid receptors. Ca 2+ Acknowledgments: This work was supported by Ministerio de Ciencia y Tecnología (MCyT) Grant BFI and Dirección General de Investigación de la Comunidad de Madrid Grant 08.5/0008.1/2003. We thank M. Sefton for editorial assistance. References [1] Huang, C.C., Lo, S.-W. and Hsu, K.-S. 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